Functional Analysis of MicroRNA Genes during C. elegans development

MicroRNAs are indispensable regulators of gene expression that are required for animal development and physiology. In addition, microRNAs have been implicated in a wide spectrum of human diseases, notably cardiovascular disease, neurodegenerative disease, diabetes, and cancer. The identification of biological functions of individual microRNAs is vital to understand their role in animal development as well as in human disease.

The ability to appropriately regulate gene expression is central to the normal development and function of cells, tissues and organisms. Small, non-coding, ~22 nucleotide RNAs, termed microRNAs, are now recognized as critical regulators of diverse cellular processes including cell proliferation, differentiation, and apoptosis. Post-transcriptional regulation of gene expression by microRNAs is essential for animal development. However, in C. elegans, most individual microRNAs are individually dispensable; worms carrying mutations in microRNA genes develop essentially normally. A critical gap in our knowledge is the identification of the specific biological pathways and direct target mRNAs regulated by miRNAs. As an eminently genetically tractable animal, C. elegans provides an ideal model system in which to study the functions of microRNAs in eukaryotes, particularly as many microRNAs show complete or near-complete conservation between worms and humans.

Specifically, work in my lab is focused on the following three areas:

1) Identification of pathways and targets regulated by microRNAs. To identify novel functions for microRNAs in C. elegans, we used a genetic approach to identify mutant phenotypes associated with the loss of individual miRNAs, possibly owing to functional redundancy between divergent miRNAs. We first examined loss of function miRNA mutations in a genetically sensitized background that has reduced overall activity of miRNAs. For this sensitized background, we used a mutation in the alg-1 gene, which encodes an Argonaute protein that functions in the miRNA pathway. We also examined loss of function miRNA mutations in a genetically sensitized background that has reduced activities of a wide array of regulatory pathways. Using these two approaches, we identified phenotypes associated with the loss of 25 out of the 31 microRNAs analyzed. Ongoing work in the lab is focused on identifying the pathways and direct targets regulated by these microRNAs.

2) Analysis of microRNA regulation of rhythmic behaviors in C. elegans. Rhythmic behaviors are ubiquitous phenomena that are observed across animal and plant phyla. These include such biological processes as smooth muscle contractions in the gut, pumping of the heart, and the release of hormones in animals as well as controlled movements of leaves and petals, and transport of solutes in plants. Three easily observable rhythms in the nematode C. elegans, pharyngeal pumping, defecation, and ovulation, allow for genetic dissection of the regulatory mechanisms that control the frequency and robustness of rhythmic behaviors. These three rhythms are all regulated by inositol 1,4,5-trisphosphate (IP3)-mediated release of intracellular calcium. A spike in calcium triggers a wave of calcium throughout the intestine that act to initiate and, importantly, to coordinate the steps of the defecation motor program every ~50 seconds. However, it remains largely unknown how cells establish and maintain the robust pattern of intercellular waves of calcium to control rhythmic behaviors. We have identified a microRNA that is expressed in the intestine and in the somatic gonad and is required for the regular execution of the defecation motor program. We hypothesize that microRNA repression of key regulatory targets acts to ensure robust expression of calcium-dependent rhythmic behaviors in C. elegans. Ongoing work in the lab is focused on characterizing the effects of the loss of this microRNA and the identification of downstream targets to define the biological activity of this microRNA.

3) Analysis of the lin-4 family member, mir-237, in developmental timing. MicroRNAs were first identified as regulators of developmental timing in C. elegans. Regulation of embryonic and post-embryonic development (larval stages L1-L4) requires the coordinated specification of cell fates in time and space. Mutations in “heterochronic” genes cause certain cells to adopt fates normally associated with earlier or later times in development, relative to stable temporal landmarks such as progression through the molting cycle. The lin-4 and let-7 microRNAs are critical regulators of temporal patterning decisions during early and late larval development, respectively. The let-7 family members, mir-48, mir-84 and mir-241, function together to regulate developmental timing decisions and regulate the temporal transition from the second to the third larval stage (L2-to-L3) through the regulation of a downstream effector, hunchback-like 1 (hbl-1). Work in the lab is looking at the function of the lin-4 family member, mir-237, in the regulation of early developmental transitions.